Acetylcholine is the primary transmitter released by enteric excitatory motor neurons. It plays a central role in the control of motility of the gastrointestinal tract. The excitatory input is received by G protein-coupled muscarinic acetylcholine receptors (mAChRs) expressed in postjunctional cells - smooth muscle cells and interstitial cells of Cajal. Most visceral smooth muscles co-express both type 2 and type 3 mAChRs (M2R and M3R). Interactions between M2R and M3R are central to understand cholinergic transmission and contraction of gastrointestinal smooth muscles. In the smooth muscle cells, co-stimulation of M2R and M3R by muscarinic agonists activates a cation current, mICAT. Recent studies demonstrate that mICAT is mainly mediated by canonical transient receptor potential 4 (TRPC4). We have shown that like mICAT, the activation of TRPC4 channel is dependent on the co-stimulation of both Gq/11 and Gi/o-coupled receptors. The TRPC4 currents share many biophysical properties and regulatory features with mICAT. The goal of the proposed project is to use TRPC4 as the molecular model to examine two unique and outstanding features concerning the activation mechanisms of mICAT and their implications in gastrointestinal smooth muscle physiology. The first is the codependence on Gq/11 and Gi/o signaling pathways for channel activation and the second is the dual regulation by intracellular Ca2+. This goal is consistent with our long-term objective in elucidating the regulatory mechanisms and physiological functions of TRP channels. We hypothesize that the two G protein signaling pathways work synergistically on TRPC4 channel to activate mICAT. Gi/o proteins act via physical interaction of the G1i/o or G23 subunits, or both, with the TRPC4 protein, and Ca2+ exerts multiple regulatory actions through calmodulin binding at distinct sites of the channel molecule. The project has two specific aims: 1) to determine the physiological significance and molecular mechanism of Gi/o-mediated mICAT activation; 2) to dissect the molecular mechanisms of regulation of mICAT (TRPC4) by intracellular Ca2+. A multidisciplinary approach that combines molecular biology (heterologous expression and site-directed mutagenesis), biochemistry (protein-protein interactions), electrophysiology (whole-cell and single channel recordings), and genetic approaches (transgenic mice that express defined TRPC isoforms and mutant channels) will be used to accomplish the proposed research. The study will enhance our understanding on excitation-contraction coupling and other contractile functions of smooth muscles and shed light on the pathogenesis and new treatment of a wide range of human diseases caused by smooth muscle dysfunctions, such as inflammatory bowel disease, irritable bowel syndrome, and urge incontinence. The molecular details of TRPC4 regulation to be generated will also significantly impact our knowledge in other physiological systems, where TRPC4 and related TRPC5 channels are known to involve in functions such as vasoconstriction/relaxation, synaptic transmission, neurite outgrowth/neural development, and learning. PUBLIC HEALTH RELEVANCE: This project focuses on the molecular mechanism of activation and regulation of muscarinic acetylcholine receptor-evoked cation current found in intestinal smooth muscle cells. The study is aimed to provide a better understanding on how neurotransmitters trigger membrane depolarization and the subsequent intracellular calcium increase to cause smooth muscle contraction in the gastrointestinal system. This will shed light on the pathogenesis and new treatment of a wide range of human diseases caused by smooth muscle dysfunctions, such as inflammatory bowel disease, irritable bowel syndrome, and urge incontinence.